The immune system functions as the body's natural defense mechanism for identifying and killing or eliminating disease-causing pathogens, such as bacteria, viruses, or other foreign microorganisms. However, with regard to cancer, including lymphomas, the immune system's natural defense mechanism is believed to be largely thwarted by natural immune system mechanisms which seek to protect “self-cells” from attack. In humans, the primary disease fighting function of the immune system is carried out by white blood cells (“leukocytes”), which mediate two types of immune responses: innate immunity and adaptive immunity. Innate immunity refers to the broad first-line immune defense that recognizes and eliminates certain pathogens prior to the initiation of a more specific adaptive immune response. While the cells of the innate immune system provide a first line of defense, they cannot always eliminate or recognize infectious organisms. In some cases, new infections may not always be recognized or detected by the innate immune system. In these cases, the adaptive immune response has evolved to provide a highly-specific and versatile means of defense which also provides long-lasting protection (immune memory) against subsequent re-infection by the same pathogen. This adaptive immune response facilitates the use of preventative vaccines that protect against viral and bacterial infections such as measles, polio, diphtheria, and tetanus.
Adaptive immunity is mediated by a subset of white blood cells (lymphocytes), which are divided into two types: B-cells and T-cells. In the bloodstream, B-cells and T-cells recognize antigens, which are molecules that are capable of triggering a response in the immune system. The human body makes millions of different types of B-cells that circulate in the blood and lymphatic systems and perform immune surveillance. Each B-cell has a unique receptor protein (immunoglobulin) on its surface that binds to one particular antigen. Once a B-cell recognizes its specific antigen and receives additional signals from a T-helper cell, it can proliferate and become activated in order to secrete antibodies (immunoglobulins; Ig) which can neutralize the antigen and target it for destruction. T-cells may also recognize antigens on foreign cells, whereby they can promote the activation of other white blood cells or initiate destruction of the targeted cells directly. A person's B-cells and T-cells can collectively recognize a wide variety of antigens, but each individual B-cell or T-cell will recognize only one specific antigen. Consequently, in each person's bloodstream, only a relatively few lymphocytes will recognize the same antigen. In this way, the complex repertoire of immune receptors generated by B and T cells enables recognition of diverse threats to the host.
Since B-cell cancers such as non-Hodgkin's lymphoma (“NHL”) are tumors arising from a single malignant transformed B-cell, the tumor cells in NHL maintain on their surface the original malignant B-cell's immunoglobulin (collectively referred to as the “tumor idiotype”) that is distinct from those found on normal B cells. The tumor idiotype maintained on the surface of each B-cell lymphoma can be used as the tumor-specific antigen for autologous idiotype cancer vaccines.
Since idiotype vaccines are individually manufactured from a tissue biopsy obtained from a patient's own tumor, they are described as personalized vaccines. This approach makes use of the fact that the unique tumor idiotype is expressed exclusively on the cancerous B-cells. So, when a full, high-fidelity copy of the idiotype is used as a vaccine, it can effectively mount a highly-specific anti-lymphoma attack that “trains” the body's own immune system to solely recognize the idiotype as a “foreign invader”, thus stimulating and recruiting the patient's own immune system to destroy micro-pockets of cancer cells that may remain following chemotherapy and potentially target and destroy newly arising lymphoma cells, thus delaying or preventing cancer recurrence.
In many cases, including in NHL, cancer cells produce molecules known as tumor-associated antigens, which may or may not be present in normal cells but may be over-produced in cancer cells. T-cells and B-cells have receptors on their surfaces that enable them to recognize the tumor associated antigens. While cancer cells may naturally trigger a B- or T-cell-based immune response during the initial appearance of the disease, this response may be only weakly specific or attenuated in such a way that it does not fully eradicate all tumor cells. Subsequently, tumor cells gradually evolve and escape from this weak immune response and are able to grow into larger tumors. In addition, because cancer cells arise from normal tissue cells, they are often able to exploit or increase existing immune tolerance mechanisms to suppress the body's immune response which would normally destroy them. In other cases, chemotherapy or other treatment regimens used to treat the cancer may themselves weaken the immune response and render it unable to reject and kill tumor cells. Even with an activated immune system; however, the number and size of tumors can often overwhelm the immune system.
B-cell and T-cell antigen receptors with diverse binding activities are generated by genomic rearrangement of variable (V), diversity (D), and joining (J) gene segments separated by highly variable junction regions. Advanced sequencing methods have recently been used to analyze B cell receptor diversity. A recent study using deep sequencing of clonal IgH (Ig heavy chain) receptor genes in chronic lymphocytic leukemia revealed unexpected intraclonal heterogeneity in a subset of cases (Campbell P J et al., “Subclonal phylogenetic structures in cancer revealed by ultra-deep sequencing,” Proc. Natl. Acad. Sci. U.S.A., 2008, 105:13081-13086). Time- and labor-intensive multi-parameter flow cytometry or custom-designed patient- and clonal-specific real-time PCR assays have been used for detection of more subtle clonal populations (Sayala H A et al., “Minimal residual disease assessment in chronic lymphocytic leukaemia,” Best Pract. Res. Clin. Haematol., 2007, 20:499-512; Ladetto M et al., “Real-time polymerase chain reaction of immunoglobulin rearrangements for quantitative evaluation of minimal residual disease in multiple myeloma, Biol. Blood Marrow Transplant., 2000, 6:241-253; Rawstron A C et al., “International standardized approach for flow cytometric residual disease monitoring in chronic lymphocytic leukaemia,” Leukemia, 2007, 21:956-964).
Assessment of lymphocyte clonality in human specimens was carried out in a population-based epidemiological study which showed that small amplified B-cell populations can be seen in almost all individuals who go on to develop chronic lymphocytic leukemia (Landgren O et al., “B-cell clones as early markers for chronic lymphocytic leukemia,” N. Engl. J. Med., 2009, 360:659-667). Detection and analysis of immune receptor clonality and evolution has been undertaken in normal and pathogenic immune reactions (Pinna D et al., “Clonal dissection of the human memory B-cell repertoire following infection and vaccination,” Eur. J. Immuno., 2009, 39:1260-1270; Wardemann H et al., “B-cell self-tolerance in humans,” Adv. Immunol., 2007, 95:83-110, each incorporated herein by reference).
Using a bar-coding strategy to achieve pooling of multiple libraries of rearranged IgH V-D-J gene loci from many human blood samples, high-throughput pyrosequencing was performed to characterize the B cell populations in a series of human clinical specimens from cancer patients and healthy people to examine the diversity of their B-cells (Parameswaran P et al., “A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing,” 2007, Nucleic Acids Res., 35:e130, incorporated herein by reference). From healthy individuals, the authors were able to estimate the normal complexity of the B-cell repertoire. With samples from the cancer patients, they obtained disease-specific signatures of clonal B-cell proliferation events. The two distinct V-D-J rearrangements in a lymph node from one cancer patient indicated that there were two separate clonal B-cell populations in the specimen, which was supported by the morphological and immunophenotypic evidence of two different B-cell lymphomas (follicular lymphoma and small lymphocytic lymphoma) in the tissue (Boyd S D et al., “Measurement and clinical monitoring of human lymphocyte clonality by massively parallel V-D-J pyrosequencing,” 2009, Sci Transmed, 1(12):1-8, incorporated herein by reference). Intratumor heterogeneity can lead to tumor evolution and adaptation, resulting in underestimation of the tumor genomic complexity. This may pose a major challenge to personalized medicine strategies for B-cell cancers.